目的 探究根皮素通过抑制丙酮酸脱氢酶激酶1（pyruvate dehydrogenase kinase 1，PDK1）-磷酸化丙酮酸脱氢酶E1亚基α 1（phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1，p-PDHA1）进而影响谷氨酰胺（glutamine，Gln）代谢介导的前列腺癌的作用机制。方法 采用不同剂量根皮素（25、50、100 μmol/L）干预人前列腺癌PC-3、DU145、LNCaP细胞与人前列腺RWPE-1细胞，采用细胞计数试剂盒（cell counting kit-8，CCK-8）测定细胞活力。将人前列腺癌PC-3细胞分为对照（二甲基亚砜，dimethyl sulfoxide，DMSO）组、根皮素（100 μmol/L）组及顺铂（0.03 mmol/L）组，Transwell法检测细胞侵袭能力、TUNEL法检测细胞凋亡水平。根皮素及Gln单独使用及联合干预PC-3细胞，试剂盒测定Gln消耗水平、谷氨酸与腺嘌呤核苷三磷酸（adenosine triphosphate，ATP）产生水平，Western blotting法测定谷氨酰胺酶1（glutaminase 1，GLS1）蛋白表达水平，同时测定细胞增殖、侵袭、凋亡等细胞生物学行为变化。利用网络药理学及生物信息学分析根皮素、前列腺癌与Gln代谢相关基因的交集。Western blotting法测定各组细胞PDK1蛋白表达水平。将PC-3细胞分为空载体对照（pcDNA3.1）组、PDK1过表达载体（pcDNA3.1-PDK1）组、PDK1敲减载体（KD-PDK1）组及其对照（KD-Control）组、PDHA1过表达载体（pcDNA3.1-PDHA1）组、KD-PDK1＋pcDNA3.1-PDHA1组及其对照KD-PDK1＋pcDNA3.1组，以及根皮素（100 μmol/L）＋pcDNA3.1-PDK1组及其对照根皮素＋pcDNA3.1组，测定各组细胞增殖、侵袭、凋亡变化、Gln消耗水平、谷氨酸与ATP产生水平及GLS1蛋白表达水平。构建前列腺癌移植瘤小鼠模型，通过根皮素干预治疗，以顺铂作为阳性对照，探究根皮素对体内肿瘤生长的影响。结果 根皮素对人前列腺RWPE-1细胞活力无显著影响，但100 μmol/L根皮素可显著抑制人前列腺癌PC-3、DU145、LNCaP细胞增殖（P＜0.05）。与对照组比较，根皮素组细胞增殖与侵袭能力显著降低（P＜0.05）、凋亡水平显著增加（P＜0.01），Gln消耗水平、谷氨酸和ATP产生水平显著降低（P＜0.05）、GLS1蛋白表达水平显著下降（P＜0.05）；Gln干预后可逆转上述结果，且根皮素与Gln联合干预PC-3细胞时，根皮素能够抑制Gln的作用（P＜0.05）。网络药理学与生物信息分析表明，PDK1为根皮素通过Gln代谢途径治疗前列腺癌的关键靶点之一，且PDK1在PC-3细胞中高表达，根皮素可显著抑制PDK1的表达（P＜0.05）。与pcDNA3.1组比较，进一步过表达PC-3细胞中的PDK1能够促进细胞增殖与侵袭（P＜0.001）、抑制细胞凋亡（P＜0.001），增强细胞中的Gln代谢（P＜0.05）。与根皮素＋pcDNA3.1组比较，过表达PDK1能够部分逆转根皮素对PC-3细胞生物学行为及Gln代谢的影响（P＜0.05）。此外，与KD-Control组比较，敲减PC-3细胞中的PDK1有助于抑制细胞增殖与侵袭、促进细胞凋亡，减弱Gln代谢水平，然而与KD-PDK1＋pcDNA3.1组比较，联合过表达PDHA1则能够逆转这一结果（P＜0.05）。体内实验表明，根皮素能够显著抑制肿瘤生长（P＜0.05）。结论 根皮素通过抑制PDK1-p-PDHA1轴进而影响Gln代谢介导的前列腺癌。

Objective To explore whether phloretin can affect the biological behavior of prostate cancer cells mediated by glutaminolysis by inhibiting pyruvate dehydrogenase kinase 1 (PDK1)-phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1 (p-PDHA1). Methods Human prostate cancer cell lines PC-3, DU145, LNCaP, and human prostate epithelial cell line RWPE-1 were treated with different concentrations of phloretin (25, 50, 100 μmol/L), and cell proliferation was assessed using the cell counting kit-8 (CCK-8). PC-3 cells were divided into control (0.1% DMSO), phloretin (100 μmol/L), and cisplatin (DDP, 0.03 mmol/L) groups, and cell invasion was detected using the Transwell method while cell apoptosis was detected using the TUNEL method. PC-3 cells were treated with phloretin and/or glutamine (Gln) separately or in combination, and Gln levels, glutamate and adenosine triphosphate (ATP) production levels were measured using kits, while glutaminase 1 (GLS1) protein expression was determined by Western blotting in cell proliferation, invasion, and apoptosis were also assessed. Network pharmacology and bioinformatics were used to analyze the intersection of phloretin, prostate cancer, and Gln-related genes. Western blotting was used to measure PDK1 protein expression levels in RWPE-1, PC-3, and PC-3 + phloretin groups. Furthermore, The PC-3 cells were divided into control pcDNA3.1 group, pcDNA3.1-PDK1 group, the KD-PDK1 group and its control KD-Control group, pcDNA3.1-PDHA1 group, KD-PDK1 + pcDNA3.1-PDHA1 group and its control KD-PDK1 + pcDNA3.1 group, and phloretin + pcDNA3.1-PDK1 group and its control phloretin + pcDNA3.1 group, Changes in cell proliferation, invasion, apoptosis, Gln levels, glutamate and ATP production levels, and GLS1 protein expression in each group were measured. A prostate cancer xenograft mouse model was established, and phloretin intervention therapy was administered, with DDP treatment serving as a positive control, to investigate the effect of phloretin on tumor growth in vivo.Results Phloretin had no significant effect on the proliferation of RWPE-1 cells but significantly inhibited the proliferation of PC-3, DU145, and LNCaP cells, with the most significant effect on PC-3 cells at a concentration of 100 μmol/L (P < 0.05). Compared with control group, the phloretin group showed significantly reduced cell proliferation and invasion ability (P < 0.05), increased apoptosis levels (P < 0.05), decreased Gln levels, reduced glutamate and ATP production levels (P < 0.05), and weakened GLS1 protein expression (P < 0.05). Gln intervention yielded opposite results, and when phloretin and Gln were co-administered to PC-3 cells, phloretin inhibited the Gln-induced effects (P < 0.05). Network pharmacology and bioinformatics analysis indicated that PDK1 is one of the key targets for phloretin in the treatment of prostate cancer through the Gln metabolism pathway. Moreover, PDK1 was highly expressed in PC-3 cells and was inhibited by phloretin (P < 0.05). Compared with pcDNA3.1 group, overexpression of PDK1 in PC-3 cells promoted cell proliferation and invasion (P < 0.05), inhibited cell apoptosis (P < 0.05), but enhanced Gln metabolism in cells (P < 0.05). Compared with the phloretin + pcDNA3.1 group, overexpression of PDK1 partially reversed the effects of phlorizin on the biological behavior and Gln metabolism of PC-3 cells (P < 0.05). Additionally, compared with the KD-Control group, knockdown of PDK1 in PC-3 cells contributed to the inhibition of cell proliferation and invasion, promotion of cell apoptosis, and reduction of Gln metabolism levels. However, compared with the KD-PDK1 + pcDNA3.1 group, co-overexpression of PDHA1 reversed these effects (P < 0.05). In vivo experiments showed that phloretin significantly inhibited tumor growth in mice (P < 0.05). Conclusion Phloretin affects Gln metabolism-mediated prostate cancer progression by inhibiting the PDK1-PDHA1 axis.

R285.5

天津市科技计划项目（20JCQNJC00550）